Although chemotherapy has been responsible for curing many people of cancer in the latter half of the 20th century, there still remain a large number of patients whose tumours either show little response to treatment, or respond initially only to recur later. For these patients, the current treatments are clearly inadequate.
The majority of the deaths arising from cancers of solid tissues can be ascribed to the process of metastasis wherein cancer cells spread from the site of origin to distant sites in the body. For example, in breast cancer, cancer cells break off from the primary tumour and metastasise via lymphatic and blood vessels. The initial spread of cancer cells is usually to the local lymph nodes, most frequently to the adjacent axilla. Thereafter breast cancer cells can disseminate via the blood stream resulting in distant metastases. These distant metastases are usually the cause of death in the majority of breast cancer patients. Therefore, anti-cancer agents that prevent metastasis would be highly desirable.
There has been substantial investment in researching the mechanisms used by normal eukaryotic cells to control progress through the cell cycle in the hope that this would lead to an understanding of how cancer arises and suggest possible targets for cancer therapy. It is currently understood that progress through the phases of the cell cycle is controlled by a class of enzymes termed “Cyclin Dependent Kinases” (CDKs). Cyclin dependent kinases are serine/threonine cyclin-dependent kinases that are synthesised continuously and maintain relatively constant levels in the cell. They are inactive on their own. CDKs are activated upon binding to their cyclin partner (De Bondt et al. (1993) Nature, 363: 595-602; Jeffrey et al. (1995) Nature, 376: 313-320), and upon phosphorylation by a CAK (cyclin activating kinase; Grana and Reddy (1995) Oncogene, 11: 211-219). CDK kinase activity can be inhibited by removal of the kinase's cyclin partner and by an inhibitory phosphorylation of a tyrosine residue close to the N-terminus of the protein. For example, CDK4 protein is activated by phosphorylation of threonine 164 but inhibited by phosphorylation of tyrosine 17. The activity of a CDK/cyclin holoenzyme can be regulated by cyclin dependent kinase inhibitors (E1-Deiry et al. (1993) Cell, 75: 817-825; Harper et al (1993) Cell, 75: 805-816; Xiong et al. (1993) Nature, 366: 701-704; Polyak et al. (1994), Genes Dev. 8: 9-22; Serrano et al. (1993) Nature 366: 704-707; Serrano et al. (1995) Science, 267: 249-252).
The cell cycle has several checkpoints to ensure that a cell does not replicate its DNA or divide under inappropriate conditions (Hartwell and Weinert (1989) Science, 296: 629-634). Before passing through these checkpoints, a cell must meet certain criteria. Molecular pathways signaling the presence or absence of these criteria influence the decision to cross the checkpoint by affecting activation of the CDK/cyclin holoenzyme responsible for regulating passage through the checkpoint. For example, positive signal transduction pathways relaying signals from cell surface receptors, such as the Ras/Raf/Erk pathway have been demonstrated to influence pRb phosphorylation, through an effect on the cyclin/CDK holoenzymes regulating the G1/S checkpoint (Lloyd et al., (1997) Genes Dev. 11: 663-677). Conversely cyclin dependent kinase inhibitors (CDKIs) such as the p16INK4, p27KIP1 and p21CIP1/WAF1 ene products can arrest cells at the G1/S checkpoint by inhibiting G1 cyclin/CDK holoenzymes. The p21WAF1/CIP1 gene may be transcriptionally activated by p53 protein providing a mechanism by which p53 protein can arrest normal cells at the G1/S checkpoint (Li et al., (1994) Oncogene, 9: 2261-2268).
Once activated, the CDK/cyclin holoenzyme initiates the events needed for the cell to enter the next phase of the cell cycle. Different CDK/cyclin holoenzymes regulate different checkpoints in the cell cycle (Hunter and Pines (1994) Cell, 79: 573-582; Sherr (1994) Cell 79: 551-555). The initiation of progress from G1 to S phase that occurs when quiescent mammalian cells are stimulated to divide by the presence of growth factors, involves the interaction of the cyclin D family with either the CDK4 gene product or the CDK6 gene product depending on the cell type (Matushime et al. (1994) Mol. Cell Biol. 14: 2066-2076; Mayerson and Harlow (1994) Mol. Cell Biol. 14: 2077-2086). In mammals, other checkpoints are controlled by different CDK/cyclin holoenzymes e.g. late G1/S is regulated by CDK2/cyclin E, progress through S phase by CDK2/cyclin A and late S/G2 by CDK1/cyclin A (Jeffrey et al. (1995) Nature 376: 313-320). Transit from G2 to mitosis is controlled by the CDK1/cyclin B complex in both mammals and yeast (Draetta (1990) Trends Biochem. Sci. 15: 378-382; Murray (1992) Nature, 359: 599-604).
Activation of CDK4 is understood to initiate transit from G1 to S phase. FIG. 2 provides a schematic diagram showing the role of CDK4 at the G1/S transition in normal cells. Activated CDK4 is thought to mediate its effects through phosphorylation of pRb and related proteins p107 and p130. In their hypophosphorylated state pRb, p107 and p130 bind E2F transcription factors. However, upon phosphorylation of pRb, p107 and p130, E2F transcription factors are released (Hijmans et al. (1995). Mol. Cell Biol. 15: 3082-3089). The free E2Fs form heterodimers with the proteins DP-1/DP-2. The E2F/DP heterodimers then bind to DNA and activate transcription of factors required for DNA synthesis (Wu et al. (1995) Mol. Cell Biol. 2536-2546). In addition, free E2F protein upregulates genes controlling cell division such as cyclin E, cyclin A, CDK1 and E2Fs. Overexpression of some members of the E2F family, such as E2F-1, however, does not only promote increased cell division, but can also lead to apoptosis. Adenoviral-mediated transfer of exogenous DNA vectors overexpressing E2F-1 to human colonic adenocarcinoma (Draus et al., (2001), Exp. Mol. Med. 33: 209-219), oesophageal (Yang et al., (2000) Clin. Cancer Res. 6: 1579-1589), melanoma (Dong et al., (1999) Cancer 86: 2021-2033, glioma (Fueyo et al., (1998) Nat. Med. 4: 685-690), breast, ovarian (Hunt et al., (1997) Cancer Res., 57: 4722-4726 and head and neck (Liu et al., (1999) Cancer Gene Ther. 6: 163-171) cancer cell lines induced apoptosis in these cell lines. E2F-1 overexpression caused cells to enter S-phase prematurely and to accumulate in G2/M from which they subsequently exited by undergoing apoptosis.
During carcinogenesis, it is currently thought that normal cells become immortalised as a consequence of disruption of the positive and negative cell signalling pathways and cell cycle control mechanisms described above, for example, amplification and overexpression of cyclins and CDKs. Amplification and overexpression of cyclin D protein occurs in many human tumours (Lammie et al., (1991) Oncogene 6: 439-444, Jiang et al, (1993) Proc. Natl. Acad. Sci USA 90: 9026-9030, Schurring et al, (1992) Oncogene, 7: 355-361, Bartkova et al., (1995) Oncogene, 10: 775-778) and cell lines (Buckley et al., (1993) Oncogene, 8: 2127-2133, Warenius et al., (1996) Int. J. Cancer 67: 224-231. Unscheduled expression of cyclin BI and cyclin E in inappropriate phases of the cell cycle has also been reported in several leukaemic and solid tumour cell lines (Gong et al, (1994) Cancer Res. 54: 4285-4288). 20-fold amplification of genomic CDK4 DNA with accompanying increases in mRNA expression has been detected in 13.8% of a series of 29 human gliomas (He et al., (1994) Cancer Res. 53: 5535-5541). Similar increases in CDK4 genomic DNA and mRNA have been found in 2 out of 14 human sarcomas (Khatib et al., (1993) Cancer Res. 53: 5535-5541).
Abnormalities in CDK inihibitors particularly mutations and altered expression of p16INK4 may also occur (Nobori et al., 1994, Okamoto et al., 1994 Jen et al., 1994). High levels of p16INK4 protein have been found to correlate with functional inactivation of the retinoblastoma gene product (Tam et al., (1994) Cancer Res. 54: 5816-5820) whilst overexpression of the CDK4 gene product has been suggested to provide an alternative mechanism to p16INK4 gene homozygous deletion.
In summary, it is believed that cancers may arise through an evolutionary process, selecting cells with gene mutations that provide a growth advantage (Ilyas et al. Eur. J. Cancer (1999) 35:335-351). By this means the normal diploid cell is progressively transformed into a fully-fledged cancer cell. Studies of early events in carcinogenesis have revealed several genetic lesions causing errors in the cell division and death pathways (Hanahan and Weinberg, Cell (2000) 100:57-70). Approximately three to seven separate molecular lesions are believed to be required to transform a normal diploid cell into a cancer cell (The Genetic Basis of Human Cancer (1999) Vogelstein and Kinzler, eds.).
A more advanced model of carcinogenesis has been proposed by Dr. Bernard Weinstein. Dr. Weinstein postulates that only certain patterns of gene expression enable cells to survive and replicate. Furthermore, it is postulated that the disruption of gene products that normally control cell division and death in early carcinogenesis results in the cell circuitry becoming “unbalanced”. In order for the cancer cell to survive and divide, certain other genes may need to become upregulated. This model may explain why gene products that normally act to inhibit cell division (such as p27KIP1 and pRb) are upregulated in certain cancer cells.
Cancer cells may metastasise from the site of origin to distant sites in the body. The protein (Kuukasjarvi et al. Cancer Res. (1997) 57:1597-1604) and mRNA populations (Hashimoto et al. Cancer Res. (1996) 56: 5266-5271) of metastatic and non-metastatic breast cancer cells have been compared in order to identify metastagenes; genes that are differently expressed in metastatic, relative to non-metastatic cells. Metastagenes code for proteins that contribute only to the potentially fatal metastatic spread of cancer cells. They do not contribute to uncontrolled growth or immortalisation. Metastagene products include enzymes such as proteases (Duff Clin. Exp. Metastasis (1992) 10:145-155), proteins associated with cell adhesion (Iwamura et al. Cancer Res. (1997) 57: 1206-1212) and motility factors (Cajot et al. Cancer Res. (1997) 57: 2593-2597; Meyer-Siegler and Hudson Urology (1996) 48: 448-452). An example of a protein thought to be associated with adhesion is osteopontin (Oates et al. Oncogene (1996) 13: 97-104, Oates et al. Cancer Metastasis Rev. (1997) 17: 1-15; Chen et al, Oncogene (1997) 14: 1581-1588) and one associated with motility is a regulatory calcium ion binding protein, p9ka (S100A4) (Barraclough et al. Eur. J. Biochem (1982) 129: 335-341; Barraclough et al. Nucleic Acids Res. (1984) 21:8097-8114; Ebralidze et al. Genes Dev. (1989) 3: 1086-1093; Gibbs et al. J. Biol. Chem. (1994) 269: 18992-18999).
Based on the present models of cancer and metastasis, attempts have been made at rational drug development. It has been considered that the gene products that are disrupted during carcinogenesis are likely to provide highly specific targets for cancer chemotherapy. Using the targets identified by this approach, new therapeutic agents are now being introduced into the clinic. These include Herceptin, which targets the her/neu cell surface receptor in breast cancer (Sliwkowski et al. (1999) Semin Oncol. 4 suppl.12: 60-70; Baselga Eur J. Cancer (2001) 37 suppl. 1:18-24), farnesyl transferase inhibitors which target the ras oncogene (Adjei et al. (2000) Cancer Research 60:1871-1877), ONYX015 (an E1B deletion mutant adenovirus), designed to target cancer cells with non-functional TP53 (Nemunaitis et al. (2000) Cancer Research 60:6359-6366), ST1571, designed to target the translocated abelson kinase in chronic myeloid leukaemia (Mauro and Druker (2001) Oncologist 6:233-238), and flavopiridol which inhibits the kinase activity of the CDK4 gene product.
Thus, the majority of targets for rational anti-cancer drug development available at present have been defined by studies of early carcinogenesis. However, in contrast to cells studied in early carcinogenesis, profound chromosomal damage can be found in the cell exhibiting the “full malignant phenotype”. The full malignant phenotype is found in clinically advanced tumours. It is characterised by an enormous diversity of structural chromosome damage (The Genetic Basis of Human Cancer (1998) Vogelstein and Kinzler eds.; Mitelman et al (1997) Nature Genet 15:417-474). In addition, cells having the full malignant phenotype also have widespread changes in gene expression when compared to normal cells (Hough et al. (2000), Cancer Research 60: 6281-6287; Waghray et al (2001) Cancer Res. 61: 4283-4286; Wang et al. (2000) Oncogene 19:1519-1528). Given the number of genetic lesions producing molecular abnormalities within the a cancer cell having the full malignant phenotype, it seems unlikely that the cell can simply be returned to its pre-cancerous, normal diploid phenotype by selectively targeting and inhibiting these abnormal early cancer genes.
There are numerous problems associated with the current approach to rational anti-cancer drug development. These include multiplicity of potential drug targets, tumour heterogeneity and genetic instability.
The number of potential drug targets available to the current approach to rational anti-cancer drug development is large and growing. At present there is no way of telling which of the many abnormal genes and gene products present in a cell having the full malignant phenotype are ultimately likely to prove the most effective drug targets.
The difficulties with rational drug development against selected molecular targets are compounded by tumour heterogeneity. Tumour heterogeneity may result from the random chaotic nature of cell division in clinical tumours. It describes the situation where tumour cells of apparently the same type in different patients, behave differently and show differences in phenotypic expression of gene products including those implicated in the process of carcinogenesis (Shackney and Shankey (1995) Cytometry 21:2-5; Harada et al. (1998) Cancer Research 58-4694-4700). As a result of these differences in the phenotypic expression of gene products, apparently similar tumour cells in different patients may respond differently to a particular anticancer drug with some cells being sensitive whilst others are resistant. This type of drug resistance is called intrinsic resistance.
Even within the same tumour in an individual patient, all cells may not exhibit the same pattern of gene expression so that some cells may be resistant to a certain type of chemotherapy to which others are sensitive. Thus chemotherapy may initially cause tumour shrinkage by killing the sensitive cells but fail to kill the resistant cells. The remaining resistant cells continue dividing to produce a cancer that is now wholly drug resistant. This process is termed acquired resistance.
Therefore, tumour heterogeneity leads to intrinsic resistance and acquired resistance of cancers to new anticancer drugs. This may account for the relatively poor clinical response rates in phase I/II studies of new anticancer agents targeting molecules disrupted in early carcinogenesis. In fact, in one completed phase II study of flavopiridol in patients with advanced gastric cancer, no clinical responses were observed (Kaubisch et al. (2000) The Cancer Journal 6: 192-210)
Genetic instability is found in the majority of cancers, if not all. It results in new mutations occurring throughout the life of a tumour (Genetic Instability in Cancer (1996) Lindahl ed.; Lengauer et al. (1998) Nature 396: 643-649). Certain of these mutations may confer drug resistance to the cells in which they occur. Genetic instability, with its ongoing molecular changes is a further cause of acquired drug resistance and makes human tumours a moving target for the rational design of chemotherapeutic agents.
Therefore, currently available treatments are not always adequate to deal with all cancers. Furthermore, agents directed to gene products involved in early carcinogenesis are unlikely to prevent metastasis.